www.nature.com/scientificreports OPEN High resolution analysis of rare copy number variants in patients with autism spectrum disorder from Received: 20 April 2017 Accepted: 4 September 2017 Taiwan Published: xx xx xxxx Chia-Hsiang Chen1,2, Hsin-I. Chen3, Wei-Hsien Chien4, Ling-Hui Li5, Yu-Yu Wu1, Yen-Nan Chiu3, Wen-Che Tsai3 & Susan Shur-Fen Gau 3,6,7 Rare genomic copy number variations (CNVs) (frequency <1%) contribute a part to the genetic underpinnings of autism spectrum disorders (ASD). The study aimed to understand the scope of rare CNV in Taiwanese patients with ASD. We conducted a genome-wide CNV screening of 335 ASD patients (299 males, 36 females) from Taiwan using Afymetrix Genome-Wide Human SNP Array 6.0 and compared the incidence of rare CNV with that of 1093 control subjects (525 males, 568 females). We found a signifcantly increased global burden of rare CNVs in the ASD group compared to the controls as a whole or when the rare CNVs were classifed by the size and types of CNV. Further analysis confrmed the presence of several rare CNVs at regions strongly associated with ASD as reported in the literature in our sample. Additionally, we detected several new private pathogenic CNVs in our samples and fve patients carrying two pathogenic CNVs. Our data indicate that rare genomic CNVs contribute a part to the genetic landscape of our ASD patients. These CNVs are highly heterogeneous, and the clinical interpretation of the pathogenic CNVs of ASD is not straightforward in consideration of the incomplete penetrance, varied expressivity, and individual genetic background. Autism spectrum disorder (ASD) represents a group of childhood-onset neurodevelopmental disorders char- acterized by abnormal social interactions, impaired verbal and nonverbal communication, and the presence of restricted interests and repetitive behaviors with long-term persistence of core features and functional impair- ment1,2. Te prevalence of ASD is various across diferent regions with an increasing trend over the years3–5 and with male excess in a male-to-female ratio of approximately 5:16–8. In the USA, the prevalence of ASD increased in the past decade according to the report of Centers for Disease Control and Prevention of USA8. It was esti- mated that around 1 in 68 persons aged eight years in the USA in 2010 was afected with ASD8. However, the increasing trend of ASD prevalence was not observed in the UK9. Te estimated prevalence of ASD in Chinese population ranged from 2.8 to 29.5 per 10,000 according to a recent review that summarized the fndings in Chinese population from several areas3. Te prevalence of ASD in Taiwan is approximately 0.3% based on the analysis of national health insurance research dataset10 and 1% based on the most recent Taiwan’s national sur- vey of child and adolescent mental disorders11 with a male: female ratio of approximately 4: 110. Due to its high prevalence, long-term impairment resulting in a great impact on individuals, families, and society12,13 and strong evidence of genetic components in its etiology14, this severe developmental disorder has been prioritized for molecular genetic studies15. Te heritability estimate of ASD is greater than 90%, attesting that genetic factors play a major role in the pathogenesis of ASD16–18. However, the genetics of ASD is very complex. Several genome-wide association stud- ies (GWAS) have identifed some common single nucleotide polymorphisms (SNPs) associated with the risk of 1Department of Psychiatry, Chang Gung Memorial Hospital-Linkou, Taoyuan, Taiwan. 2Department and Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan. 3Department of Psychiatry, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan. 4Department of Occupational Therapy, College of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan. 5Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. 6Graduate Institute of Brain and Mind Sciences, National Taiwan University, Taipei, Taiwan. 7Graduate Institute of Epidemiology and Preventive Medicine, National Taiwan University, Taipei, Taiwan. Correspondence and requests for materials should be addressed to S.S.-F.G. (email: [email protected]) SCIENTIFIC REPORTS | 7: 11919 | DOI:10.1038/s41598-017-12081-4 1 www.nature.com/scientificreports/ CNV size ASD (n = 335) Controls (n = 1093) CNV rate ASD/CON Likelihood Ratio Chi-square P* <100 Kb 333 394 0.99/0.36 109.68 <0.0001 100–400 Kb 88 43 0.26/0.04 101.02 <0.0001 Deletion >400 Kb 18 14 0.05/0.01 15.41 <0.0001 Total 439 451 1.31/0.41 163.23 <0.0001 <100 Kb 263 229 0.79/0.21 146.25 <0.0001 100–400 Kb 150 144 0.45/0.13 84.17 <0.0001 Duplication >400 Kb 55 19 0.16/0.02 80.21 <0.0001 Total 468 392 1.40/0.36 224.09 <0.0001 <100 Kb 596 623 1.78/0.57 188.03 <0.0001 100–400 Kb 238 187 0.71/0.17 153.20 <0.0001 Deletion and Duplication >400 Kb 73 33 0.22/0.03 89.69 <0.0001 Total 907 843 2.71/0.77 273.33 <0.0001 Table 1. Comparisons of rare autosomal CNVs in patients with autism spectrum disorders and control subjects. P < 0.005 was considered statistically signifcant as it was corrected by 12 using Bonferroni test. ASD, such as common variants on 20p12.1, 5p14.1, 1p13.219–23. Tese common SNPs, however, have only small efects on autism ASD risk, and of note, few if any of these SNPs were replicated in diferent studies. Furthermore, accumulating evidence suggests that rare genetic and genomic mutations also contribute to the genetics of ASD24. Conventional cytogenetic studies of ASD have revealed a variety of rare chromosomal abnormalities associated with ASD25–28 indicating aberrant genomic rearrangements are part of the genetic mechanism of ASD. Notably, the recent advent of array-based comparative genomic hybridization (aCGH) technology has discovered various submicroscopic copy number variations (CNVs) of genomic DNA associated with ASD29,30, leading further sup- port to the idea that ASD is a genomic disorder in a subset of the patients. Tese ASD-associated CNVs are usu- ally individually unique and of low frequency, but together they account for approximately 5–10% of idiopathic ASD29, hence, constituting a part of the genetic architecture of ASD22,31,32. Te discovery of genomic mutations in ASD-associated CNVs not only helps decipher the genetic complexity of ASD23, but also helps shed some light on the neurobiology and pathogenesis of ASD32–37. In our previous studies, we reported four pathogenic CNVs in certain ASD patients38,39, indicating that CNVs also play a role in the genetic architecture of ASD in our patients. To have a better understanding of the scope of rare genomic CNVs in our ASD patient population, we recruited a sample of more than 300 ASD patients and conducted a genome-wide CNV screening in this sample. Results Clinical characteristics. A total of 335 (95.7%) out of 350 cases and 1093 (98.4%) out of 1111 controls passed a series of quality control of CNV experiments. We investigated the ethnicity of cases and controls by performing principle component analysis (PCA) with SNP genotype data from all the participants of this study and the individuals included in HapMap study. Te results demonstrated that the cases and controls are clustered together with the Han Chinese (Supplementary Figure 1). Terefore, the ethnicity of the participants of this study was confrmed to be the Han Chinese. Further, all the CNV data were subjected to the burden analysis. Te patient group consisted of 299 boys and 36 girls with the mean age of 9.4 ± 4.0 years, while the control group consisted of 525 males and 568 females with the mean age of 68.1 ± 10.1 years. Te ADI-R (Autism Diagnostic Interview-Revised) interviews revealed that the 335 patients scored 20.43 ± 6.12 in the “qualitative abnormal- ities in reciprocal social interaction”, 14.75 ± 4.32 in the “qualitative abnormalities in communication, verbal”, 8.19 ± 3.33 in the “qualitative abnormalities in communication, nonverbal”, and 6.95 ± 2.47 in the “restricted, repetitive and stereotyped patterns of behaviors.” All the participants with ASD were noted to have had abnormal development at or before 36 months of age. Teir current average intelligence quotients (IQ) were 94.85 ± 22.55 (range, 40 to 148) for full-scale IQ, 96.74 ± 2.04 (range, 41 to 145) for performance IQ, and 95.08 ± 23.79 (range, 44 to 148) for verbal IQ. Among the 335 ASD patients, nine had been diagnosed with epilepsy (3.04%), four had been suspected of seizure (1.35%), and 19 had ever had a febrile convulsion (6.42%). Tese data are also provided in the Supplementary Table 1. CNV fndings. Te rates of rare CNV (<1% in the patients) at autosomes and X-chromosome were exam- ined between the patient and control groups. CNV regions on autosomes were analyzed in all samples while CNV regions on sex chromosomes were analyzed in male samples only. We found a signifcant excess of the overall rate of rare CNV at autosomes in the ASD patients (2.71) compared with the control subjects (0.77). Te over-representation of rare autosomal CNV rate in ASD was still present when the rare CNVs were grouped into deletion and duplication or classifed according to the size as <100 kb, 100–400 kb, and >400 kb (Table 1). In the analysis of rare CNV at X-chromosome, we compared only the rate of rare CNV between male patients and male control subjects.